Mechanisms of mitochondrial transcription

描述(申请人提供)：线粒体基因表达的遗传或年龄相关缺陷可降低或消除线粒体功能，并导致多种人类病理，包括神经退行性疾病和心血管疾病、糖尿病和癌症。线粒体转录是基因表达的关键过程，相应地，这些致病改变中的一些与参与转录的蛋白质有关。然而，我们对线粒体转录机制的不同组成部分的功能了解相对较差。表征线粒体转录的分子机制将使我们能够理解这一过程中微小的遗传或环境变化如何导致基因表达缺陷和线粒体病理的发展。这项建议旨在提供对线粒体转录过程的功能洞察，并研究存在于转录因子中的rRNA甲基转移酶活性的重要性。我们的目标是揭示转录启动的机制，转录与核糖体生物发生之间的联系，以及转录缺陷与线粒体疾病的关系。该建议有三个目的：(I)研究与16S rRNA甲基转移酶相关的两个转录因子TFB1M和TFB2M之间的结构和功能差异，并研究它们与其核酸底物相互作用的特异性；(Ii)研究TFB1M和TFB2M的甲基转移酶活性，它们在转录和核糖体生物发生中的重要性，以及它们与母系遗传性耳聋的关系；(Iii)确定转录启动的机制，转录过程中所需的蛋白质-蛋白质相互作用和转录机制的结构。该提案将利用大量的初步数据，包括两个新的晶体结构，发现对启动至关重要的新相互作用，以及开发新的方法来研究体外rRNA甲基转移酶的活性。我们将应用各种分子遗传学、生化和生物物理技术来研究转录所必需的功能相互作用以及TFB1M和TFB2M的酶活性。电子显微镜、小角X射线散射和X射线结晶学将被用来提供额外的结构洞察，并反过来促进我们对该过程的功能理解。预期的结果将极大地增加我们对线粒体转录、转录因子的个体作用、转录与核糖体生物发生之间的关系以及母系遗传性耳聋的分子基础的了解。最终，提高我们对转录过程的机械性理解将有助于阐明转录、线粒体功能障碍和疾病之间的关系。

DESCRIPTION (provided by applicant): Genetic or age-related defects in mitochondrial gene expression can reduce or eliminate mitochondrial function and cause multiple human pathologies, including neurodegenerative and cardiovascular disease, diabetes and cancer. Mitochondrial transcription is a key process for gene expression, and accordingly some of these pathogenic alterations have been associated with proteins involved in transcription. However, our functional understanding of the different components of the transcription machinery in mitochondria is relatively poor. Characterizing the molecular mechanisms underlying mitochondrial transcription will allow us to understand how small genetic or environmental alterations in this process can result in gene expression deficiencies and the development of mitochondrial pathology. This proposal aims to provide functional insight into the process of mitochondrial transcription and investigate the importance of the rRNA methyltransferase activities present in transcription factors. We aim to unravel the mechanism of transcription initiation, the connections between transcription and ribosome biogenesis and the relationship between transcriptional defects and mitochondrial disease. The proposal has three aims: (i) to investigate the structural and functional differences between two transcription factors related to 16S rRNA methyltransferases, TFB1M and TFB2M, and to study the specificity of their interaction with their nucleic acid substrates; (ii) to investigate the methyltransferase activitie of TFB1M and TFB2M, their importance for transcription and ribosome biogenesis and their relationship with maternally inherited deafness; (iii) to determine the mechanisms of transcription initiation, the protein-protein interactions necessary for the process and the architecture of the transcription machinery. The proposal will take advantage of substantial preliminary data, including two novel crystal structures, the discovery of a novel interaction critical for initiation and the development of new methods to study the rRNA methyltransferase activities in vitro. We will apply various molecular genetics, biochemical and biophysical techniques to investigate the functional interactions necessary for transcription and the enzymatic activities of TFB1M and TFB2M. Electron microscopy, small angle x-ray scattering and X-ray crystallography will be used to provide additional structural insight and, in turn, facilitate our functional understanding of the process. The expected results will dramatically increase our knowledge of mitochondrial transcription, the individual roles of transcription factors, the relationships between transcription and ribosome biogenesis and the molecular basis of maternally inherited deafness. Ultimately, improving our mechanistic understanding of the transcription process will help clarify the relationship between transcription, mitochondrial dysfunction and disease.